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Transcript
Evaluation of living Gumboro vaccineprepared from locally variant isolated strain for
controlling of IBD problem in Egypt
*Susan,
S. El-mahdy, ** Manal. A. Afify, **Abd Alim, G., and *Helal, A. M.
(*) Central Lab. For Evaluation of Veterinary Biologics (CLEVB)
(**) Faculty of Vet.Medicine, Cairo Uni.
[email protected]
Abstract:
For controlling of Gumboro problem in Egypt; the locally isolated variant strain was adapted on
BGM-70 cell line used for preparation of tissue culture living vaccine. The IBDV had a titer of 107.5
TCID50/ml (104.5 TCID50 per dose) after five passages in BGM-70 cell culture. Evaluation of prepared
living variant vaccine was measured by identity,purity, safety and potency. The result revealed that the
prepared vaccine was free from bacterial (aerobic-anaerobic), fungal mycoplasmal and extraneous viral
contamination. The protection percentage was 92%, 96% and 100% in groups vaccinated with imported
vaccine compared to 96% in group vaccinated with local prepared vaccine when challenged with
vvIBDV. On the other hand, the protection percentage with significant higher in groups challenged with
IBD variant strain. Finally, protective dose fifty for locally prepared variant vaccine were significant in
full and half dose.
Keywords: Local variant Infectious bursal disease virus (IBDV)-Tissue culture living vaccine(T.C)
Introduction:
Gumboro disease (IBD) was described as a specific new disease by Cosgrove (1962) and was
referred to as “avian nephrosis”. Later, it was termed as infectious bursal disease by Hitchner (1970). It
is acute highly contagious viral disease of young chickens characterized by enlargement of the bursa of
Fabricius and sever renal damages (Ivanyi and Morris, 1976). Acute IBD emerged in 1980s (Jackwood,
et al., 1982). IBD was first reported in Egyptian flocks in the early seventies (El-Sergany et al., 1974).
However interest in IBDV antigenic characterization was triggered by the appearance of the very virulent
IBD in vaccinated Egyptian flocks (El-Batrawi and El-Kady 1990;Khafagy, et al.,1991).Several reports
have classified the Egyptian IBDV isolates as classical IBDV (Khafagy, et al., 1991; Bekhit, 1996;
Ibrahim, 2000). Presently, evidence of circulating variant IBDV strains were isolated from flocks
vaccinated using classical IBDV vaccines (El-Khiat, 2003; Hussein et al., 2003 and Metwally et al.,
2003).Variant strains of IBDV are usually isolated from vaccinated flocks. These IBDV variants are
antigenically different from classic strains of IBDV; as it is devoid the classical epitope(s) defined by
neutralizing monoclonal antibodies (Snyder et al., 1988b; Snyder, 1990; Snyder et al., 1992).Most of
these epitopes are located in the VP2 hyper variable region (Snyder et al., 1988a). Immunization of
chickens is the principle method used for the control of IBD in chickens. There are many choice of
available live vaccine based on virulence as intermediate virulence and highly attenuated strains while
virulent vaccine not available commercially up till now, also the vaccines can be classified according to
antigenic variation. The vaccine must be safe, pure and efficient (Tsukamoto et al., 1995).Inspire of
vaccination against IBD, some flocks were suffered from immunosuppression .As well as some flocks up
to 3weeks (unsusceptible age of classical IBD) were immunosuppressed with atrophied bursae and lesion
of proventriculus. The aim of this study was carried out to controlling IBD problem in Egypt by
preparation of T.C vaccine from the isolated variant strain and possibility of cross protection between
variant and classical one.
Material and Methods:
Viral strains:
1. Infectious bursal disease viruses (IBDV):
1.1-Field isolated variant viruses:
Infectious bursal disease virus isolate Egy-IBD Var 2009 VP2 gene, partial cds Submitted in gen bank at
Accession No. : JN 118617.
-1-
1.2-Challenge(very virulent (VVIBD)and classic) viruses:
The viruses used in the challenge were in form of infectious allantoic fluid, they were isolated from field
cases and identified by phylogenic analysis .They were titrated in SPF ECE as described by Villegas
(1990) with titer103.5 EID50/ml and it is calculated according to the method of Reed and Muench (1938).
2. Newcastle disease viruses (NDV):
2.1-Newcastle disease challenged virus:
It is a virulent virus of Newcastle disease of field isolate, it was obtained from the Newcastle Disease
Department, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo (VSVRI), Its infectivity
titer was106 EID50/ml.It used for challenge the experimental chicks 3 weeks post vaccination.
2.2-Newcastle diseaseheamagglutinating antigen:
Propagation of LaSota strain NDV in embryonated chicken egg was applied according to Allan et al.,
(1972) for HI test. ND Heamagglutinating antigen was adjusted 4 HA unite.
3. Vaccine strains:
3.1- Infectious bursal disease (IBD)vaccines:
Four living attenuated imported gumboro vaccine were used:
 Intermediate plus obtained from CEVA with batch no. 3412T3S, A CevaIBD2; with
titer105.5 EID50 / dose.
 Intermediate obtained from IZO with batch no. 0018E IZO Gumboro 2,with titer 105.5
EID50 / dose
 Intermediate obtained from Intervet International B.V.,Box Meer, Holland with batch no.
09614HMol NobilisGumboro 228E;with titer 105.5 EID50 / dose
 Classical D78obtained from Hipra with batch no.9R2y-2 M.G. Hipra, with titer 105.2
EID50 / dose.
3.2- Newcastle Disease vaccine: (vaccination of experimental chicks for evaluation of
immunosuppression effect of IBD vaccine)
NDV Hitchner B1 vaccinal strain obtained from Intervet B.V., Box Meer Holland with
batch no. 08811EJ01 Nobilis ND Hitchner. This vaccine used for immunosuppression
study of IBD with titer 107.5EID50/ml.
4. Experimental hosts:
4.1. Experimental chicks:
One day old specific pathogen free (SPF) chicks were used in this study. All birds
were reared in separated cages and kept in strictly hygienic measures. The room was
previously cleaned, thoroughly disinfected and were provided with autoclaved
commercial water and feed. The chicks used for evaluation of prepared vaccine study.
4.2. Specific pathogen free (SPF) embryonating chicken eggs (ECE):
It was obtained from the SPF production farm, Koum Oshein, El-Fayoum, Egypt. Eggs
were kept in the egg incubator at 37 °C with humidity 40-60% used according to Allan et al., (
1972) for detection of extraneous haemagglutinating agents in prepared vaccine.
4.3. Experimental mice:
Swiss male mice were obtained from (CLEVB), used for studying the Safety of T.C IBD
vaccine for mammalian species. (Ecotoxicity test)
5. Tissue cultures and cell culture media: (Lennelte, 1964)
Primary chicken embryo fibroblast cell (CEF) was obtained from (CLEVB); which prepared as
-2-
described by Schat and Purchase (1989). Trypsin-version solution prepared according to Lennelte
(1964); Hank's balanced salt solution (HBSS) prepared according to Hank andwallance (1949);
Minimum Essential Medium (MEM) was prepared according to the manufacturer’s instructions; and
Bovine serum was mycoplasma free and virus screened "Gibco Limited, Scotland, and UK". The method
used for inoculation in the microtitre plates was done according to Rossiter and Jessett (1982) Tissue
culture used for detection of extraneous agents in prepared IBD vaccine (Avian Lymphoid Leukosis)
according to 9 CFR Ch. 1 (1-1-09 Edition) 11.30 (2009).
Baby Grivet Monkey Kidney cell line 70 (BGM-70) were used forvariant virus propagation and titration
according to the method described by Jackwood et al., (1986). It was obtained from Vacciera,
Agouza,Giza
6. Serum samples:
Sera were collected from the tested chickens three weeks after experimental infection (post
inoculation) and for the evaluation of the immune response of both prepared and imported IBD vaccine
stored at -20°C till used.
Methods:
1. Propagation of local field isolates in BGM-70 cell lines: According to Jackwood et al. (1986).
2. Enzyme Linked Immuno-Sorbent Assay (ELISA): (Snyder et al.,1986).
ELISA Kit was obtained from Symbiotic Corporation 11011 VIA Forntera San Digo, CA 92127,
U.S.;Leukosis ELISA Kit with Batch No. FS 5254 and Infactiousbursal kit with Batch no FS5155.ELISA
Reader :Micro plate reader USA,VERSA max,with serial number was B02274.
3. Polymerase Chain reaction (PCR): (Council of Europe, 1999).
PCR used for test of extraneous agents in IBD prepared vaccine according to Council of Europe
(1999)and for detect of Identity of IBD Vaccine by using DNA Star analysis; RNA extraction Kit using
Bioflux simply total RNA extraction Kit Cat # BSC 52 S1 ). DNA extraction Kit using Bioflux Mega Bio
virus DNA purification kit cat # BSC 12 S14. Amplification by using BIOER Reverse transcription
polymerase chain reaction (RT–PCR) kit one step cat # BSBO 7 MI for (infectious bronchitis,, TRT and
Avian Influenza) as extraneous RNA agents in IBD variant vaccine. Amplification by using Ferments
DreamTaq green PCR Master Mix Cat # K 1084 for (ILT, DH, Fowl Pox and Marek's disease virus) as
extraneous DNA agents in IBD variant vaccine .The amplicone was subjected to sequencing
4. Haemaulutmation test (HA): (Majujabe and Hitchner ,1977)
It carried out according to the standard procedure described by Majujabe and Hitchner(1977) to
detect of extraneous haemagglutinating agents inprepared IBD vaccine according to 9 CFR Ch. 1. (1-109) Edition 113 -3 3 (2009).
5. Haemagglutination inhibition (HI) test for NDV:
The test was carried out according to the standard procedure described by MajujabeandHitchner
(1977) for the haemagglutinating activity of NDV antigen was an essential primary procedure using the
HA test (Anon, 1971) to determine HA unites used in HI test
6. Titration of local field isolates in BGM-70 cell lines:
This method was carried out according to Pedro and Graham (1980), this technique used
for evaluation of the potency of tissue culture adopted live IBDV vaccines. The TCID50was calculated
according to Reed and Muench (1938)
-3-
7. Evaluation of bursal lesion:
a.Organ/body weight ration:
.It was carried out according to Sharma et al.,(1989):Collected bursae; spleen;orproventruclus were
weighed and the organ/body weight ratio was determined as follows:
Organ weight (gm)
Organ/body weight ratio=
Body weight (gm)
b. Estimation of bursal weight index:
x 1000
It was carried out according to Lucio and Hitchner(1979).
B:BW ratio of the inoculated group
Bursal weight index= Mean of B:BW ratio of the non-inoculated control group
Chickens with bursal index lower than 0.7 were considered to have bursal atrophy
Results:
Preparation of living T.C. IBD vaccine from local variant strain:
Adaptation of virus in BGM-70:
The local variant strain was adapted on BGM-70 cell line and for preparation of living vaccine.In
table (1), the results showed that BGM-70 cell line was satisfactory as it yielded good cytopathic changes
in BGM-70 cell started after 7 days post inoculation (P.I) in the first passage, while the maximal
development of CPE was achieved at 2 days P.I. in the fifth passage. The observed cytopathic changes
were characterized by a marked granulation of cell cytoplasm, cell rounding, cell aggregation, loss of
adherence of cells and formation of cell syncytia. The IBDV had a titre of 10 7.5 TCID50/ml (104.5
TCID50/ dose) after adaptation on BGM-70 cell culture.
Quality control of prepared living variant T.C (IBD) vaccine:
Bacterial and fungal sterility:
The results of living T.C IBD vaccine is free from bacterial and fungal contamination.
Mycoplasmic sterility
The results of T.C IBD vaccine is free from mycoplasma
Identity:
Polymerase chain Reaction (PCR) by using primer specific to infectious bursal virus Visualization of
the PCR products on a 1 % agarose gel, DNA band was seen at 300bp .
Purity tests:
The T.C IBD vaccine does not contain any extraneous agent either haemagglutinating agent inoculation of 9 – 10
day old embryonated SPF eggs; primary culture of chicken embryofibroblasts (Test for avian leucosis virus) or by using
PCR test for on other extraneous viral agents.
Virus titration:
After adaption virus was tittered by serial passages on BGM-70 cell line the virus titer increased in
the higher passage than the lower ones as seen in table (1) .The virus titer reached 7.5 log10 TCID50 after
five passages in BGM-70 cell line
Safety of prepared vaccine:
-4-
Ten field doses of locally prepared variant T.C. vaccine and imported classical D78 one are
administered by eye drop to each of 15 SPF one day old chickens; respectively as groups 1&2 while
another 15 SPF chickens were kept as control, all chickens were kept under observation for 3 weeks for
any systemic reactions, and post-mortem examinations. Five chickens on each group at7th, 14th day post
vaccination. Bursae of Fabricius of chickens were examined macroscopically for evidence of any
changes due to IBDV infection
All vaccinated chickens had no change or show notable clinical signs or macroscopic lesions that
are attributable to the vaccine with comparison the negative control chickens.
Safety in mammalian species (Ecotoxicitytest): Thirty Swiss mice were divided into three groups (10/
group) as follow:
Group (1): vaccinated group receive the T.C IBD vaccine with 10 x dose intra peritoneal, and group (2):
receive imported classical D78; Group (3): non-vaccinated controlled group.
T.C IBD vaccine proved to safe for mammalian species as there was no mortality occurred in any group,
nor general or local reaction. Growth was identical in the all groups during the period of observation. .
Results of safety test in mammalian species (Ecotoxicity lest) gave no evidence for abdominal toxicity in Swiss mice
inoculated intra peritoneal with 10 X dose of T.C IBD vaccine.
Evaluation of organ/body weight ratio.
.Forty SPF chicks were divided into 2groups for studding the effect of the prepared living TCIBD variant
vaccine on organs/body weight in relation to control birds.
Evaluation of the immunosuppressive effect of the prepared living T.C.IBD vaccine.
Fifty SPF one chicks were used for measuring the possible immunosuppression of prepared variant IBD
vaccine by measuring HI titer against ND vaccine compared with control one.
Potency test and cross protection:
Three hundred and fifty SPF one day old chickens were divided into equally 2 groups (A&B)
into 7 subgroups as in tables (4). Groups from (1- 4) vaccinated via eye drops with different imported
vaccines (CEVA, IZO, IntervetandHipra, respectively) and group (5) was vaccinated with locally
prepared T.C variant vaccine. While groups (6&7) kept as control group (6) control +ve for vvIBD, in
subgroup (A) and for variant local isolate in subgroup (B) while group 7 control –ve for all experiment.
Chickens were kept under observation for 3 weeks post vaccination and the serum samples were
collected from all groups. The immune response was determined in vivo by challenging birds with
103.5EID50 / dose virulent IBD strain for all vaccinated groups except group (7) in subgroups (A) and
challenged with variant local isolate with dose 103.5EID50 in subgroups (B) and in vitro by measuring
ELISA titer post-vaccination and their mean antibody titer that shown in table (4).
Detection of protective dose fifty for prepared vaccine:
One hundred and twenty five , one day old SPF chicks used for this experiment and divided into (5)
groups as following : group 1,2,3and 4vaccinated via eye drop route by full,0.5, 0.25and 0.125 of dose
respectively while group 5 kept as positive control . Control negative non-vaccinated non-challenge group
of 5 chickens was also included for this experiment as group (6). All groups were kept under observation
daily. At 3 weeks post vaccination the serum samples were collected from all groups .The immune
response was determined by ELISA test and challenging the 1st five groups with known vvIBDV with
titer 103.5 EID50 / dose
The calculation of PD50:
-5-
D = 50 - protection %of dose less than 50%
Protection% more than 50%-protection% of dose less than 50%
Y =
Dose over 50%
Dose less than 50
X = d x log y
Discussion
We designated our trails for the local variant strain preparation of T.C. vaccine compared with
living IBD vaccines that used in the field as method of prevention the economic losses and the
immunosuppressive effect of subclinical form of IBD.
The locally prepared living variant T.C. IBD vaccine on BGM-70 cell gave good results for
propagation and titration 7.5 log 10 TCID50 / ml, the harvested and subjected as described by Hassan
and Saif (1996) and Hassan et al., (1996) , and our results agreed with the result obtained by Lee and
Lukert (1986) as they attempted isolation of IBDV from turkeys and chickens as well as from samples of
challenge strains received from other laboratories . Turkey strain (five of five) were readily adapted to
CEF cells after 3 to 10 blind passages , only two of nine chicken strains could be grown only in chicken
embryo fibroblast cells, and with that of Saif (1984) who used BGM-70 cells successfully for isolation of
IBDV from the bursa of naturally infected chickens;.The result of titration was judged according to the
parameters of code of Federal Regulation USA "Part 133. 331-9 CFR.1 (2008) in which IBDV titers
must be not less than 103.5 TCID50 / dose. So the prepared TC IBD Variant strain was Satisfactory with
104.5 TCID50/dose. Lukert and Davis (1974) who successfully adapted wild type IBDV from infected
bursa to grow in cell derived from the chicken embryo bursa which considered as nonimmunosuppressive vaccine.According to the parameters of Code of Federal Regulation, USA, Part 133331-9 CFR Ch. 1, 1-1-97 Ed"(2008) in which IBDV titer must be notless than 3.5 log10 EID50/ml, the
intermediate vaccine lost its ability to beprotective by 10 weeks and 12 weeks at 37°C and 25°C ;
respectively. While the mild vaccine lost its ability to be a protective vaccine by the 6 th week and 10th
week al 37°C and 25°C;respectively.
The above mentioned results were agreed with Fekria et al., (1993) who reported a
reduction in virus titer equal l0 6.3 log10 EID50 for 6 months at room temperature..The results of
sterility, safety, potency and immunogenicity of the local variant IBDV was done according to
Federal regulation USA "part 133. 331 – 9CFR ch.1, 1-1-97 ED"(2008); Giambrone and
Closser (1990) and Ismail and Saif (1991). It is free from bacterial fungal and Mycoplasma
which judged according to parameters of European pharmacopoeia (1998) in which the vaccine
must be sterile and free from any contamination. The TCIBDV does not contain any extraneous
agent eitherhaemagglutinating agent; avian leucosis virus or any another extraneous, viral agents
when use SPF inoculation, tissue calture; ELISA test or PCR. These results was judged
according to 9CFR (2009), Europian pharmacopeia (1989 and 1998) , British pharmacopeia
(1990) and GRIMV (1992). PCR used for detect of identity of TCIBDV as described in OIE
(2010)All experimental chicks had no changes or show notable clinical signs or macroscopic
lesions that are attributable to the vaccine in chick’s vaccination with 10X field dose in
comparison with the negative control chicks.Theirbursae of Fabricius are examined
macroscopically for evidence of any changes due to IBDV. Our results for ecotoxicity test according
to European pharmacopeia (1997).So that TCIBD vaccine which locally prepared from variant isolate
was safe and sterile.
Bursal indexes in vaccinated SPF groups were significantly higher than the challenge controls
(table-2). The TCIBD locally prepared variant vaccine protected against bursal damage as indicated by
significantly lower bursal lesions in vaccinated birds (Perozo,et al., 2009), the bursae from chickens with
bursal/body weight index higher than 0.7 found to be histologically normal (Lucio and Hitchner 1979)
-6-
Bursa / Body weight ratio were calculated according to Sharma et al., (1989) revealed the results .
Assessment of bursal atrophy based on the criteria proposed by Lucio and Hitcliner (1979), that BI equal
to or Less than 0.7 to be considered atrophy, the vaccinated group revealed no bursal atrophy during 13
days PI. Variant infected group revealed marked, rapid-decrease in bursal size as early as 36 hours PI up
to 13 days PIas recorded in histopathology. Infected group with classic virus strain revealed an increase in
bursal size to approximately twice the size of the control group within 4 days PI, then decreased in size to
approximately half of the control group during 13 days PIBursal atrophy in this group begin to atrophied
on the 6th day PI (BI=0.53) to the end of the observation period. These findings are in agreement with the
findings of Rosenberger et al. (1987); and Tsai and Saif (1992) and Lukert and Saif (2008).Chickens
infected with local, variant T.C IBDVs had larger splenic size and SI, mostly during the observation
periods PI (from 36 hours to 14 days PI) as compared with control ones. These findings are in agreement
with Tsai and Saif (1992). According to the assessment criteria proposed by Lucio and Hitchner
(1979),that splenic index (SI), lower than the lowest control index considered atrophied, whereas an index
higher than the highest control index was considered as hypertrophied. Based on these criteria, all IBDV
infected groups showed hypertrophied spleens. These results agreed withthe findings of Resales et al.
(1989).These results correlated with histopathological findings for evaluation of the extent of splenic
damage, the picture revealed early mild lymphocytic depletion accompanied with mild edema in all
infected groups. This finding explain the hypertrophy of spleen and agreed with those reported by
Tanimura et al., (1995), who reported the ability of field and vaccinal strains of 1BDV to infect
monocytes and macrophages and enlargement of splenic reticular cells..
The locally prepared variant T.CIBD vaccine is non-immunosuppressive as shown in table(3)
Our results agree with Mazariegos et al., (1990) who studied the effect of pathogenesis of seven
commercially available IBD vaccines and immune-suppressive effects on SPF chicks. These
vaccine strains are intermediate in their pathogenicity in susceptible SPF chickens. One-day-old
and 3-day-old SPF chickens were vaccinated with these vaccines. Two weeks after IBD
vaccination, they were vaccinated with NDV. The pathogenic and immunosuppressive effects of
IBD vaccines were evaluated by the antibody response to NDV vaccination. It was found that
these strains were highly variable in their virulence and immunosuppressive properties. Three
tested strains were found to be highly virulent and immunosuppressive; two others were
moderate; and two could be classified as mild
At potency test of the prepared local variant vaccine Three hundred and fifty SPF one day old
chicks were divided equally 2 subgroups (A&B), into each group (7) subgroups as shown in tables (4).
The subgroups (A&B) from 1,2,3 and 4 in each group were vaccinated via eye drops with imported
vaccines from ( CEVA, Izo, Intervet and Hipra; respectively) while subgroup(A&B) of no. 5 was
vaccinated with locally prepared T.C. variant vaccine. At the same time subgroup 6 and 7 were consider
positive and negative control. At 3 weeks of age, serum samples were collected from all subgroups for
measuring antibody titer using ELISA.
The group (A) was challenged with vvIBD with a dose of 10 3.5EID50 and the group (B) was challenged
with variant local isolate with a dose of 103.5EID50.The birds were observed for 2 weeks post challenge
and signs, lesions and mortalities were recorded.Cross protection for the prepared vaccine gave protection
96% and 100% against vvIBD and variant one; respectively.
GMT ELISA titer 8271 when compared with imported vaccine which give 92,100,100, 96%for
intermediate plus, intermediate, 228E and classical D78; respectively with GMT ELISA titer
10317,10610,10251,11092. our results accord withKhafagy et al., (1991) who found that mortalities by
three Egyptian vvIBDV isolates in SPF chickens were 40% , 80% and 100% and with Gimbrone and
Clay (1986) and Mousa et al., (1988).
Amal (1999) showed the ELISA antibody titer was in gradual increase and reached its maximal level at
the 3rd week post vaccination and the antibody titer was higher in chicken groups vaccinated with
intermediate strain than those with mild strain vaccine. The same results recorded also by Monier
(1997).High serologic immune response as early as 36 hours PI with variant, classic and vaccinal strains
and gradually increased to high levels at the end of the observation period (49 days of age). These results,
-7-
are in partial agreement with those reported by Bayyari et al,. (1996), who recorded neutralizing
antibodies 3 days PI with standard and variant isolates while at 4 days after vaccination with an
intermediate type of vaccine, associated with complete protection after challenge Van den Berg and
meulemans( 1991), and mostly attributed to the used kits where the wells of the plates were coated with
IBDV-derived bursal homogenates.
Finally, protective dose fifty for prepared vaccine was calculated when SPF chicks were inoculated with
full, 0.5, 0.25 and 0.125 field dose then challenged with 103.5 EID50/dose of local prepared variant
vaccine. The results revealed that only 0.125 and 0.25 does was insignificant PD50, while full & half dose
were significant PD50 according to OIE (2008) that the protective dose of vaccine must be more than
90%.
From above mentioned results we can use living T.C. IBD vaccine prepared from local variant isolated
virus strain as method for control of IBD disease in Egypt. On the other hand; the subclinical for of the
disease caused by variant strains need different programmers for control. Inactivated vaccines and live
vaccine made from variant strains protected chickens from the disease caused by either variant or
standard strains, whereas inactivated vaccines made from standard strains did not protect, or only partially
protect against challenge with variant strains (Rosenberger et al., 1985, and Ismail and Saif, 1991).
Corresponding author
Susan, S. El-Mahdy
Central Lab. for Eval. of Vet. Biol. Abbassia Cairo, Egypt (CLEVB)
[email protected]
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Table (1) Propagation and titration of virus on BGM-70 cell lines showing number of
passages and time needed appearance of CPE
No. of passage
Time needed for CPE
Virus titre
1
7 days
2.5
2
6 days
3.0
3
5 days
4.5
4
3 days
6.5
5
2 days
7.5
No. : NumberCPE:cytopathiceffect
: titre of virus (log 10 TCID50 / ml)
Table (2) Evaluation of organ/body weight ratio:
No fo
Treated
SPF
Chicks
3 days
O
days
7 days
10 days
14 days
B
S
P
B
S
P
B
S
P
B
S
P
Vairant
ICIBD
20
0
*1.5
*1.3
1.0
1.4
*1.2
0.95
0.7
*0.49
1.0
0.66
0.78
0.9
Control
20
0
1
1
1
1
1
1
1
1
1
1
1
1
B: bursal/body weight ratio S: spleen /body weight ratioP: proventriculus/body weight ratio
*significant difference at p≤ 0.05
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Table (3): Results of immunosuppression test
No. of group
G1 vaccinated
G2
Indicator
G3
control +ve
G4
control -ve
No. of dead
birds/ total
number of birds
2/15
Protection
percentage
Mean HI log2
titer
86.6%
6.9
1/15
93.03%
7.2
15/15
0%
1.9
0/5
100%
1.9
The experimental chicks were fed on sterile ration and water, divided into 4 groups as follow:
G1: 15 birds vaccinated with prepared living variant T.C. vaccine at one day old via eye drop and then at
2 weeks old vaccinated with ND vaccine HB1 with titer 107.5 EID50 / dose by eye drop.
G2: 15 birds vaccinated with ND vaccine HB1 at 2 weeks old and act as indicator for NDV.
G3: 15 birds control non-vaccinated challenge group with vvNDV with titer 106.5 EID50 / dose.
G4: 5 birds control non-vaccinated non-challenge.
All groups measuring the immune response by HI test at 4 weeks old.
The locally prepared living attenuated T.C. variantstrain is consider as nonimmunosuppressivevaccine
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Table (4): Results of potency test:
No. of groups
Types of
vaccine used
GMT of
ELISA
No. of dead birds/ total
number of birds
Protective %
*
G1(A&B)
Vac1
10317
2/25
92%
G2(A&B)
G3(A&B)
G4(A&B)
Vac2
Vac3
Vac4
10610
10251
11092
0/25
0/25
1/25
100%
100%
96%
G5(A)
G5(B)
G6(A)
G6(B)
T.C.V.V
T.C.V.V
Control+ve
Control+ve
8271
8271
156
156
2/25
0/25
20/20
5/20
96%
100%
0%
80%
G7(A&B)
Control-ve
156
0/10
100%
N.B.: GMT of ELISA titer of control positive serum is equal or more than 3000
GMT: Geometric mean of ELISA antibody titer against IBDV.
A:vaccinated birds challenged with vvIBDv
B:vaccinated birds challenged with isolated variant IBD strain
Vac 1: intermediate plus CEVA vaccine with titer 105.5 EID50 / dose
Vac2: intermediate (gumboro 2) IZO with titer 105.5 EID50 / dose.
Vac3: intermediate Nobilis 228E with titer 105.5 EID50 / dose.
Vac4: classical D78 Hipra with titer 105.2 EID50 / dose.
T.C.V.V.: locally prepared T.C. variant vaccine with titer 104.5 EID50 / dose and challenged with field
isolated vvIBDV in subgroup (A) andsubgroup (B) challenged with field isolated variant IBDstrain.
G6: non vaccinated and challenged with vvIBDV in subgroup(A)and subgroup(B) challenged with field
isolated variant IBD strain
G7 (A&B): non vaccinated and non-challenged group
No clinical sings or lesion were recorded in all vaccinated group.
Birds in group (6) showed atrophied bursae with slight hemorrhages on proventriculus.
*Significant difference at P ≤ 0.05.
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Table (5): Results of PD50 of different doses for locally prepared T.C.IBD variant vaccine:
Dose
No. of
group
ELISAGMT
Protection against challenge
AntibodyTiter
Total no.
of birds
No. of
protect bird
No. of
dead bird
Protect
%
Full dose
G1
8271
25
23
2
92
0.5 dose
G2
8007
25
23
2
92
0.25 dose
G3
2610
25
22
3
88
0.125 dose
G4
2040
25
20
5
80*
Control+ve
G5
242
25
0
25
0
Control–ve
G6
242
5
5
0
100
N.B.: the protective dose for vaccine must be more than 90% according to OIE (2008).
GMT of ELISA titer of control positive serum is equal or more than 3000.
GMT = Geometric mean of ELISA antibody titer against IBDV.
*significant difference at p≤0.0
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